The studies described in this research proposal are directed at determining the biochemical mechanism and biological function of DNA helicases. The proteins to be investigated include the E. coli recBCD enzyme, rep protein, and uvrD protein. These proteins play important roles in the processes of DNA replication, recombination and DNA repair. The biochemical mechanism of helicase action will be investigated by taking advantage of a rapid, continuous, and accurate fluorescence helicase assay for DNA unwinding that has been recently developed in my laboratory. This assay poses several advantages over traditional methods and will be used to biochemically characterize the helicase activities of the recBCD enzyme, rep protein and uvrD protein. In addition, the relationship of the ATPase activity of these helicases to their DNA unwinding activity will be examined. To better understand the physical basis of the DNA unwinding activity, the equilibrium DNA and ATP binding properties of these protein will be examined. All of these studies will also be extended to a characterization of the biochemical properties of mutant DNA helicases in order to attempt to relate the biochemical activities with biological function.